Ornithine decarboxylase from calf liver. Purification and properties.
Ornithine decarboxylase (ODC) was purified 25000-fold from calf liver to apparent homogeneity by methods developed to circumvent the lability of the enzyme. Appropriate ratios of sample protein applied to column size and/or gradient size were derived for each purification procedure (ion-exchange, gel filtration ahd hydroxylapatite chromatography, electrophoresis, and thiol affinity chromatography) to maintain enzymatic activity. The enzyme was labile to dilution at all steps of the purification; the inclusion of poly(ethylene glycol) or additional protein decreased but did not eliminate the activity loss. The purified enzyme had a Stokes radius of 3.14 and a molecular weight of 54000. The Km for ornithine was 0.12 mM, and pyridoxal phosphate was 2.0 microM; the pH optimum for the decarboxylation reaction was 7.0. Analysis by sievorptive ion-exchange chromatography indicated the presence of three ionic forms. In the presence of Tris-barbital buffer containing thioglycolic acid, the ODC preparation assumed an apparent molecular weight of 100000 and a Stokes radius of 4.5 and retained full catalytic activity.
D000818:Animals; D002262:Carboxy-Lyases / Q000302:isolation & purification*; D002417:Cattle; D002614:Chelating Agents / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D004355:Drug Stability; D004492:Edetic Acid / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D009955:Ornithine Decarboxylase / Q000302:isolation & purification* / Q000378:metabolism
D002614:Chelating Agents; D004492:Edetic Acid; D002262:Carboxy-Lyases; D009955:Ornithine Decarboxylase